The present invention relates to a novel bilirubin oxidase, a process for its production and use thereof.
Bilirubin oxidase is an enzyme which catalyzes the reaction of two moles of bilirubin with one mole of oxygen yielding two moles of biliverdin and two moles of water. Various such enzymes have been reported, such as those derived from mycota of genus Myrocesium (Japanese Patent Application Laid Open No. 12032/1985), g. Bacillus (Japanese Patent Application Laid Open No. 209587/1986), g. Schizophyllum (Japanese Patent Application Laid Open No. 135886/1984), g. Coprinus, g. Trametes, g. Choriolama, g. Pholiota, g. Pleurotus, g. Rendides and g. Fusidopsis (Japanese Patent Application Laid Open No. 198971/1984) and from plantae of families Solanaceae, Musaceae and Liliaceae (Japanese Patent Application Laid Open No. 78580/1985) and so on.
Bilirubin is a yellow substance formed in blood by the decomposition of hemoglobin and constitutes a principal pigment of bile produced in liver. In blood serum, bilirubin exists in both conjugated and free forms. When the amount of conjugated bilirubin increases in blood serum, a dysfunction of the bilirubin transport system to the duodenum may occur by conjugation in the liver microsome and, if the amount of free bilirubin becomes excessive, a hemolytic anemia etc. will occur. Therefore, the ability to analytically quantify both the conjugated and the free bilirubin is clinically very important.
With respect thereto, bilirubin oxidase is also very useful for intentionally decreasing the bilirubin level in a sample to be analyzed.
Known bilirubin oxidases as mentioned above have low substrate specificity, so that their application in the field of, such as, clinical chemistry has not been without problem. Thus, all known bilirubin oxidases derived from microorganisms exhibit a substrate specificity to biliverdin, so that they are not suitable for quantitative analysis of bilirubin. On the other hand, the enzymes derived from plants suffer from restriction with regard to availability due to seasonal limitation, which is disadvantageous for industrial production, as well as with respect to a difference in the degree of substrate specificity exhibited for each specific enzyme. For example, it is suggested in the patent literature that the enzyme obtained from Solanaceae has no effect on biliverdin and hemoglobin. Known bilirubin oxidases act on substances having the tetrapyrrole structure and on phenolic substances. Therefore, the action of these oxidases would be considerably influenced by the presence of foreign substances, if employed for the biochemical examination of blood during clinical examination.